RayBio Human/Mouse/Rat Cyclic AMP Enzyme Immunoassay Kit

RayBio Human/Mouse/Rat Cyclic AMP Enzyme Immunoassay Kit Catalog #: EIA-CAMP, EIAM-CAMP, EIAR-CAMP User Manual Last revised August 7, 2017 Caution: Extraordinarily useful information enclosed ISO 13485 Certified 3607 Parkway Lane, Suite 100 Norcross, GA 30092 Tel: 1-888-494-8555 (Toll Free) or 770-729-2992, Fax:770-206-2393 Web: www.raybiotech.com, Email: info@raybiotech.com 1

Table of Contents Section Page # I. Introduction 3 II. General Description 4 III. How It Works 4 IV. Storage 5 V. Reagents 5 VI. Additional Materials Required 6 VII. Reagent Preparation A. Preparation of Plate and Anti-cAMP Antibody B. Preparation of Biotinylated Peptide (Item F) C. Preparation of Standards D. Preparation of Positive Control E. Preparation of Samples F. Preparation of Wash Buffer and HRP-Strep 6 6 7 8 9 9 10 VIII. Assay Procedure 10 IX. Assay Procedure Summary 11 X. Calculation of Results A. Typical Data B. Sensitivity C. Standard Curve Range D. Reproducibility E. Assay Diagram 12 12 12 12 12 13 XI. Specificity 14 XII. Select Publications 14 XIII. Troubleshooting Guide 15 Please read the entire manual carefully before starting your experiment 2

I. Introduction Cyclic adenosine monophosphate (camp, cyclic AMP, or 3',5'-cyclic adenosine monophosphate) is a second messenger important in many biological processes. camp is derived from adenosine triphosphate (ATP) and used for intracellular signal transduction in many different organisms, such as transferring into cells the effects of hormones like glucagon and adrenaline, which cannot pass through the plasma membrane. camp functions through three main effectors: PKA, the guaninenucleotide-exchange factor (GEF) EPAC and cyclic-nucleotide-gated ion channels. The intracellular levels of camp are regulated by the balance between the activities of two enzymes adenylyl cyclase (AC) and cyclic nucleotide phosphodiesterase (PDE). Cyclic AMP has been shown to be involved in cell growth, differentiation and general metabolism, and it is important for many biological function, especially in the cardiovascular, nervous and immune systems. The measurement of intracellular Cyclic AMP in tissues and cell cultures may help to provide a clearer understanding of the physiology and pathology of many disease states. 3

II. General Description The RayBio camp Enzyme Immunoassay (EIA) Kit is an in vitro quantitative assay for detecting camp peptide based on the competitive enzyme immunoassay principle. In this assay, a biotinylated camp peptide is spiked into the samples and standards. The samples and standards are then added to the plate, where the biotinylated camp peptide competes with endogenous (unlabeled) camp for binding to the anti-camp antibody. After a wash step, any bound biotinylated camp then interacts with horseradish peroxidase (HRP)-streptavidin, which catalyzes a color development reaction. The intensity of the colorimetric signal is directly proportional to the amount of captured biotinylated camp peptide and inversely proportional to the amount of endogenous camp in the standard or samples. A standard curve of known concentration of camp peptide can be established and the concentration of camp peptide in the samples can be calculated accordingly. III. How It Works 4

IV. Storage The entire kit may be stored at -20 C to -80 C for up to 6 months from the date of shipment. For extended storage, it is recommended to store at -80 C. Avoid repeated freeze-thaw cycles. For prepared reagent storage, see table below. V. Reagents Component EIA Microplate (Item A) Size / Description 96 wells (12 strips x 8 wells) coated with secondary antibody. Storage / Stability After Preparation 1 month at 4 C* Wash Buffer Concentrate (20X) (Item B) 25 ml of 20X concentrated solution. 1 month at 4 C Standard camp Peptide (Item C) Anti-cAMP Polyclonal Antibody (Item N) Assay Diluent D (Item K) Assay Diluent B (Item E) Biotinylated camp Peptide (Item F) HRP-Streptavidin Concentrate (Item G) Positive Control (Item M) TMB One-Step Substrate Reagent (Item H) 2 vials of Lyophilized camp Peptide. 1 vial is enough to run each standard in duplicate. 2 vials of Lyophilized anti-camp. 15 ml of 5X concentrated buffer. Diluent for standards and samples. 15 ml of 5X concentrated buffer. Diluent for anticamp antibody and HRP-Streptavidin. 2 vials of Lyophilized Biotinylated camp Peptide, 1 vial is enough to assay the whole plate. 600 µl 200X concentrated HRP-conjugated streptavidin. 1 vial of Lyophilized Positive Control. 12 ml of 3,3,5,5'-tetramethylbenzidine (TMB) in buffer solution. Do not store and reuse Do not store and reuse 1 month at 4 C 1 month at 4 C Do not store and reuse Do not store and reuse Do not store and reuse N/A Stop Solution (Item I) 8 ml of 0.2 M sulfuric acid. N/A *Return unused wells to the pouch containing desiccant pack, reseal along entire edge. 5

VI. Additional Materials Required 1. Microplate reader capable of measuring absorbance at 450 nm 2. Precision pipettes to deliver 2 µl to 1 ml volumes 3. Adjustable 1-25 ml pipettes for reagent preparation 4. 100 ml and 1 liter graduated cylinders 5. Absorbent paper 6. Distilled or deionized water 7. SigmaPlot software (or other software which can perform four-parameter logistic regression models) 8. Tubes to prepare standard or sample dilutions 9. Orbital shaker 10. Aluminum foil 11. Plastic wrap VII. Reagent Preparation Keep kit reagents on ice during reagent preparation steps. A. Preparation of Plate and Anti-cAMP Antibody 1. Equilibrate plate to room temperature before opening the sealed pouch. 2. Label removable 8-well strips as appropriate for your experiment. 3. Assay Diluent D (Item K) and 5X Assay Diluent B (Item E) should be diluted 5- fold with deionized or distilled water. 4. Briefly centrifuge the anti-camp antibody vial (Item N) and reconsititute with 55 µl of 1X Assay Diluent B to prepare the antibody concentrate. Pipette up and down to mix gently. 5. The antibody concentrate should then be diluted 100-fold with 1X Assay Diluent B. This is your anti-camp antibody working solution, which will be used in step 2 of Assay Procedure (Section VIII). Note: The following steps may be done during the antibody incubation procedure (step 2 of Assay Procedure) 6

B. Preparation of Biotinylated camp (Item F) 6. Briefly centrifuge the vial of Biotinylated camp (Item F) and reconstitute with 20 µl of ddh 2 O before use. 7. See the image below for proper preparation of Item F. Transfer the entire contents of the Item F vial into a tube containing 10 ml of 1X Assay Diluent D. This is your Working Stock of Item F. Pipette up and down to mix gently. The final concentration of biotinylated camp will be 2 ng/ml. a. Second Dilution of Item F for Standards: Add 2 ml of Working Stock Item F to 2 ml of 1X Assay Diluent D The final concentration of biotinylated camp will be 1 ng/ml. b. Second Dilution of Item F for Positive Control: Add 100 µl of Working Stock Item F to 100 µl of the prepared Positive Control (Item M). (See section D for Positive Control preparation) The final concentration of biotinylated camp will be 1 ng/ml. c. Second Dilution of Item F for samples: Add 125 µl of Working Stock Item F to 125 µl of prepared sample (see section E for sample preparation). This is a 2-fold dilution of your sample. The final concentration of biotinylated camp will be 1 ng/ml. 7

C. Preparation of Standards 8. Label 6 microtubes with the following concentrations: 1,000 ng/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml and 0 ng/ml. Pipette 450 µl of biotinylated camp Item F working solution (prepapred in step 7a) into each tube, except the 1,000 ng/ml (leave this one empty). It is very important to make sure the concentration of biotinylated camp is 1 ng/ml in all standards. 9. Briefly centrifuge the vial of camp Standard (Item C). Reconstitute with 10 µl of ddh 2 O and briefly vortex if desired. Pipette 8 µl of Item C and 792 µl of 1 ng/ml biotinylated camp working solution (prepared in step 7a) into the tube labeled 1,000 ng/ml. Mix thoroughly. This solution serves as the first standard (1,000 ng/ml camp standard, 1 ng/ml biotinylated camp). 10. To make the 100 ng/ml standard, pipette 50 µl of the 1,000 ng/ml camp standard into the tube labeled 100 ng/ml. Mix thoroughly. 11. Repeat this step with each successive concentration, preparing a dilution series as shown in the illustration below. Each time, use 450 µl of biotinylated camp and 50 µl of the prior concentration until the 0.1 ng/ml is reached. Mix each tube thoroughly before the next transfer. 8

D. Positive Control Preparation 12. Briefly centrifuge the Positive Control vial (Item M) and reconstitute with 100 µl of ddh 2 O. 13. Refer to step 7b. This is a 2-fold dilution of the Positive Control. The final concentration of biotinylated camp should still be 1 ng/ml. The Positive Control is a mouse serum sample sample that serves as a system control to verify that the kit components are working. The resulting OD will not be used in any calculations; if no positive competition is observed please contact RayBiotech Technical Support. The Positive Control may be diluted further if desired, but be sure the final concentration of biotinylated camp is 1 ng/ml. E. Sample Preparation 14. If you wish to perform a 2-fold dilution of your sample, proceed to step 7c. If you wish to perform a higher dilution of your sample, dilute your sample with 1X Assay Diluent D before performing step 7c. EXAMPLE (to make a 4-fold dilution of sample): a. Dilute sample 2-fold (62.5 µl of sample + 62.5 µl of 1X Assay Diluent D). b. Perform step 7c (125 µl of working solution Item F + 125 µl of sample prepared above). The total volume is 250 µl, enough for duplicate wells on the microplate. It is very important to make sure the final concentration of the biotinylated camp is 1 ng/ml. Note: Optimal sample dilution factors should be determined empirically, however you may contact technical support (888-494-8555; techsupport@raybiotech.com) to obtain recommended dilution factors for serum. 9

F. Preparation of Wash Buffer and HRP 15. If Item B (20X Wash Concentrate) contains visible crystals, warm to room temperature and mix gently until dissolved. 16. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1X Wash Buffer. 17. Briefly centrifuge the HRP-Streptavidin vial (Item G) before use. 18. Dilute the HRP-Streptavidin concentrate 200-fold with 1X Assay Diluent B. Note: do not use Assay Diluent D for HRP-Streptavidin preparation in step 18 VIII. Assay Procedure 1. Keep kit reagents on ice during reagent preparation steps. It is recommended that all standards and samples be run at least in duplicate. 2. Add 100 µl of Anti-cAMP Antibody (Item N) (See Reagent Preparation step 5) to each well. Incubate for 1.5 hours at room temperature with gentle shaking (1-2 cycle/sec). You may also incubate overnight at 4ºC. 3. Discard the solution and wash wells 4 times with 1X Wash Solution Buffer (200-300 µl each). Washing may be done with a multichannel pipette or an automated plate washer. Complete removal of liquid at each step is essential to good assay performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 4. Add 100 µl of each standard (see Reagent Preparation Section C), Positive Control (see Reagent Preparation Section D) and sample (see Reagent Preparation Section E) to appropriate wells. Be sure to include a blank well (Assay Diluent only). Cover wells and incubate for 2.5 hours at room temperature with gentle shaking (1-2 cycles/sec) overnight or at 4ºC. 5. Discard the solution and wash 4 times as directed in Step 3. 6. Add 100 µl of prepared HRP-Streptavidin solution (see Reagent Preparation step 18) to each well. Incubate for 45 minutes at room temperature with gentle 10

shaking. It is recommended that incubation time should not be shorter or longer than 45 minutes. 7. Discard the solution and wash 4 times as directed in Step 3. 8. Add 100 µl of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking (1-2 cycles/sec). 9. Add 50 µl of Stop Solution (Item I) to each well. Read at 450 nm immediately. IX. Assay Procedure Summary 1. Prepare all reagents, samples and standards as instructed. 2. Add 100 µl anti-camp to each well. Incubate 1.5 hours at room temperature or overnight at 4ºC. 3. Add 100 µl standard or sample to each well. Incubate 2.5 hours at room temperature or overnight at 4ºC. 4. Add 100 µl prepared Streptavidin solution. Incubate 45 minutes at room temperature. 5. Add 100 µl TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature. 6. Add 50 µl Stop Solution to each well. Read at 450 nm immediately. 11

X. Calculation of Results Calculate the mean absorbance for each set of duplicate stands, controls, and samples and subtract the blank optical density. Plot the standard curve using SigmaPlot software (or other software which can perform four-parameter logistic regression models), with standard concentration on the x-axis and percentage of absorbance (see calculation below) on the y-axis. Draw the best-fit curve through the standard points. Percentage absorbance = (B-blank OD)/(B 0 -blank OD) where B = OD of sample or standard and B 0 = OD of zero standard (total binding) A. Typical Data These standard curves are for demonstration only. A standard curve must be run with each assay. B. Sensitivity The minimum detectable concentrations of camp is 4.4 ng/ml. C. Standard Curve Range 0.1-1,000 ng/ml D. Reproducibility Intra-Assay: CV<10% Inter-Assay: CV<15% 12

E. Assay Diagram Recommended Plate Layout: 13

XI. Specificity This EIA kit is designed to only detect Cyclic AMP. XIV. Select EIA Publications 1. Plum L, Lin HV, Dutia R, Tanaka J, Aizawa KS, et al. The Obesity Susceptibility Gene Carboxypeptidase E Links FoxO1 Signaling in Hypothalamic Proopiomelanocortin Neurons with Regulation of Food Intake. Nature Med. 2009;15(10):1195-1201. (Ghrelin EIA, EIA-GHR-1) 2. Hug C, Lodish HF. Visfatin: a new adipokine. Science. 2005; 307(5708):366-7. 3. Kim MK. Crystal structure of visfatin/pre-b cell colony-enhancing factor 1/nicotinamide phosphoribosyltransferase, free and in complex with the anticancer agent FK-866. J Mol Biol. 2006; 362(1):66-77. 4. Revollo, J.R., et al. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J. Biol. Chem. 2004; 279: 50754-50763. 5. Oh-I S, Shimizu H, Satoh T, et al. Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature 2006; 443 (7112): 709-12. 6. Zhang J, Ren P, Avsian-Kretchmer O, Luo C, Rauch R, Klein C, Hsueh A. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake. Science 2005; 310 (5750): 996-9. 7. Cummings D, Weigle D, Frayo R, Breen P, Ma M, Dellinger E, Purnell J. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J Med 2002; 346 (21): 1623-30. 8. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2002; 407 (6806): 908-913.9. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999; 402 (6762): 656-60. 14

XIII. Troubleshooting Guide Problem Cause Solution Poor standard curve Inaccurate pipetting Improper standard dilution Check pipettes Briefly centrifuge Item C and dissolve the powder thoroughly by gently mixing Low signal Improper preparation of standard and/or biotinylated antibody Too brief incubation times Inadequate reagent volumes or improper dilution Briefly spin down vials before opening. Dissolve the powder thoroughly. Ensure sufficient incubation time; assay procedure step 2 may be done overnight Check pipettes and ensure correct preparation Large CV Inaccurate pipetting Air bubbles in wells Check pipettes Remove bubbles in wells High background Plate is insufficiently washed Contaminated wash buffer Review the manual for proper wash. If using a plate washer, ensure that all ports are unobstructed. Make fresh wash buffer Low sensitivity Improper storage of the ELISA kit Stop solution Follow storage recomendations in sections IV and V. Keep substrate solution protected from light. Add stop solution to each well before reading plate 15

RayBio ELISA Kits Over 2,000 ELISA kits available, visit www.raybiotech.com/elisa-kits.html for details. This product is for research use only. 2015 RayBiotech, Inc 16